H19Inhibits RNA Polymerase â…¡-mediated Transcription By Disrupting HnRNP U-actin Complex

H19is one of the earliest identified and studied long non-coding RNAs. It is presumed that H19is essential for regulating development and disease conditions. H19is associated with carcinogenesis of many type cancers and closely related to cancer pathogenesis. But the biological function and regulation mechanism of this conserved RNA, particular on transcription remain largely unknown. So studies of possible biological function and molecular machanism may provide cues for the role of H19affecting in physiological and pathological conditions. We base our study on all above-mentioned questions and hypothesis.We firstly performed the RNA pulldown experiment. Specifically, we constructed the whole H19and antisense H19on pSPT19. in vitro synthesized sense strand and antisense strand RNA and freezed if the required concentration is satisfied. Additionally, we prepared toatal protein of Hep3B cells for the final RNA pulldown. After identification of differentially expressed proteins of the sense H19and antisense H19by Mass Spectrum, it turns out that the interacting protein of H19is heterogeneous ribonucleoprotein hnRNP U. we also tested the results through western blotting and RIP. After confirming the interacting protein of H19is hnRNP U. inorder to further validate the interaction between H19and hnRNP U in our cell-based systems, we next performed deletion-mapping experiments to determine which region of H19is associated with hnRNP U. To this end, we carried out RNA pulldown experiments with truncated versions of H19followed by western blotting to detect the bound of hnRNP U. These analyses identified a882nt region at the5’ end of H19that is required for its association with hnRNP U.After confirming the association between H19and hnRNP U. we started from hnRNP U, which is a DNA/RNA binding protein to explore the physiological and pathological function and mechanism. After searching the related researches, we found that hnRNP U could act different functions if combining with different transcription factors. More over. hnRNP U is also involved in RNA Pol II transcription regulation. It has been found that the combination of hnRNP U and actin can promote transcription of RNA Pol Ⅱ CTD by promoting its phosphorylation process.Under the indication of above-mentioned researches, we wondered whether H19is also involved in RNA Pol II transcription regulation. We enhanced1119expression by transfecting an1119expression vector into Hep3B and Huh-7cells and employing the pcDNA3.1vector as the negative control. We then measured global transcription by incorporation of EU into nascent RNA transcripts that revealed specific transcription by Pol Ⅱ. Global transcription assay indicated that compared with control cells EU incorporation was decreased in Hep3B and Huh-7cells when1119was overexpressed. These results suggest that1119inhibits RNA Pol Ⅱ mediated transcription.We next tested whether H19could regulate phosphorylation situation of CID. which separately recognizes the phosphorylated Ser5in the Pol Ⅱ CTD heptad repeats. The results showed that CTD phosphorylation was inhibited in1119overexpressed Hep3B and Huh-7cells compared with control cells. In contrast, when H19and hnRNP11was meanwhile overexpressed. CTD phosphorylation was restored. This result indicates that H19inhibits RNA Pol Ⅱ CTD phosphorylation. and the observed inhibition by1119is mediated through hnRNP U.In conclusion, we analyzed the biological function of H19in HCC cells, explored the association between1119and hnRNP U and the function which1119inhibits RNA polymerase Ⅱ mediated transcription. It enlarged the researches in the function and mechanism of1119and may be helpful for the further study.